U.S. patent number 6,114,292 [Application Number 09/487,629] was granted by the patent office on 2000-09-05 for hematological analyzer sampling probe cleansing method.
This patent grant is currently assigned to Sysmex Corporation. Invention is credited to Susumu Hoshiko, Miki Miyaji.
United States Patent |
6,114,292 |
Hoshiko , et al. |
September 5, 2000 |
Hematological analyzer sampling probe cleansing method
Abstract
A method of using a cleansing composition for cleansing a
quantitatively aspirating sampling probe in an automated
hematological analyzer is disclosed. The cleansing composition
employed in the method is formulated to cleanse instantaneously on
contact, and practically eliminates carry-over of hematological
sample material, assaying reagents or of the cleansing composition
itself. The cleansing composition is an acidic aqueous solution of
pH 5.0 or less, including (1) a substance having a primary amino
group; and (2) one or more nonionic surfactants selected from the
group consisting of polyoxyethylene alkyl ether, polyoxyethylene
alkyl phenyl ether, polyoxyethylene alkyl ester and polyoxyethylene
sorbitan ester. Assay-material contaminated surfaces of the
sampling probe are contacted with a cleansing amount of the
cleansing composition.
Inventors: |
Hoshiko; Susumu (Kobe,
JP), Miyaji; Miki (Akashi, JP) |
Assignee: |
Sysmex Corporation (Kobe,
JP)
|
Family
ID: |
17456636 |
Appl.
No.: |
09/487,629 |
Filed: |
January 20, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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161672 |
Sep 29, 1998 |
6043205 |
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Foreign Application Priority Data
|
|
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|
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Oct 1, 1997 [JP] |
|
|
9-268301 |
|
Current U.S.
Class: |
510/161;
134/22.1; 134/22.11; 134/22.12; 422/44; 422/50; 422/68.1; 510/181;
510/405; 510/413; 510/421; 510/499 |
Current CPC
Class: |
C11D
1/72 (20130101); C11D 1/74 (20130101); G01N
35/1004 (20130101); C11D 3/33 (20130101); C11D
11/0041 (20130101); C11D 3/30 (20130101) |
Current International
Class: |
G01N
35/10 (20060101); C11D 001/72 (); C11D 003/30 ();
C11D 007/08 () |
Field of
Search: |
;134/22.1,22.11,22.12
;422/44,50,68.1 ;510/161,181,405,413,421,499 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Gupta; Yogendra
Assistant Examiner: Boyer; Charles
Attorney, Agent or Firm: Shinjyu An Intellectual Property
Firm
Parent Case Text
This is a division of application Ser. No. 09/161,672, filed Sep.
29, 1998, now U.S. Pat. No. 6,043,205.
Claims
What is claimed is:
1. A method for cleansing a pipette in an automated analyzer for
analyzing multiple assay items simultaneously, having
quantitatively aspirating pipettes for aspirating and dispensing
assay material aliquots including analytical samples, assaying
reagents, and reaction solutions thereof, said method
comprising:
dispensing assay material from a quantitatively aspirating
pipette;
subsequently contacting assay-material contaminated surfaces of the
pipette with a cleansing amount of a cleansing composition for
cleansing the pipette, wherein the cleansing composition is an
acidic aqueous solution of pH 5.0 or less and includes
(1) a compound having a primary amino group; and
(2) one or more nonionic surfactants selected from the group
consisting of polyoxyethylene alkyl ethers, polyoxyethylene alkyl
phenyl ethers, polyoxyethylene alkyl esters and polyoxyethylene
sorbitan esters.
2. A method as set forth in claim 1, wherein the quantitatively
aspirating pipette is in contact with said cleansing composition
for approximately 0.1 to 20 seconds.
3. A method as set forth in claim 1, wherein the quantitatively
aspirating pipette is in contact with said cleansing composition
for approximately 0.2 to 1.0 seconds.
4. A method as set forth in claim 1, wherein the quantitatively
aspirating pipette is for analysis in hemostasis and thrombosis
assays.
Description
TECHNICAL FIELD
The present invention relates to a cleansing method for use in an
automated hematological analyzer. The invention more particularly
relates to a method for cleansing a quantitatively aspirating
sampling probe in an automated hematological analyzer capable of
simultaneous, multi-item analysis.
BACKGROUND ART
Clinical analytical procedures are increasingly becoming automated
owing to technological advances, such that, for example, multiple
items can be assayed by several different reagents simultaneously
in one device. The extent of clinical monitoring items in the area
of hemostasis and thrombosis does not end merely with blood
coagulation time, but ramifies into quantitating the factors in the
clotting/fibrinolytic system, and to quantitating fibrin
degradation products; and it is now possible to make these
measurements with a single device.
Automated hematological analyzers accordingly are able to
investigate any hemostatic disorder, as well as risk of thrombosis.
That is, by performing multiple assays on hematological samples,
"coagulation profiles" are produced, from which bleeding disorders
as well as the potential for pathological formation of
cardiovascular blood clots (thrombi) can be evaluated.
Coagulation assays are performed in the hematological analyzer by
mixing test reagents into hematological samples in reaction tubes
and monitoring for diagnostic change; typically the time to onset
of clotting in the sample is measured.
In human blood coagulation, in the pathway common to both the
intrinsic (plasma-mediated) and extrinsic (tissue-mediated)
pathways, the protease Factor X (Stuart Factor) is activated, which
in turn activates prothrombin, by cleaving it to yield thrombin.
Thrombin in turn cleaves fibrinogen to yield clot-forming
fibrin.
Tissue factor (TF), released from tissues exposed to plasma from a
ruptured blood vessel, can activate Factor X directly. Accordingly,
a laboratory blood coagulation test known as the prothrombin time
(PT) assay can be conducted to evaluate the extrinsic and common
pathways. A test reagent containing tissue factor (also called
thromboplastin) is added to a plasma sample, activating Factor X
via the extrinsic route. The time to clot formation in the sample
following the addition of the thromboplastin reagent is measured as
the PT.
An evaluation of the integrity of the intrinsic pathway is by
assaying for Factor VIII (antihemophilic factor), which for example
can test for hemophilia. Factor VIII functions with other
coagulation cascade proteases and calcium ions to activate Factor
X. Accordingly, a Factor VIII reagent may be added to a plasma
sample and, as with the PT test, the time to clotting monitored,
except that therein the intrinsic pathway is evaluated.
On the other hand, clot-forming fibrin, the protein end product of
blood coagulation, must eventually be degraded, which occurs
through factors having fibrinolytic functions.
Plasmin is the activated enzyme responsible for fibrin degradation
in the fibrinolytic system, and is derived from converted
plasminogen in the blood. Plasmin activity is in turn regulated by
.alpha..sub.2
-antiplasmin, a principal inhibitor of fibrinolysis. Monitoring
plasmin levels can accordingly indicate hemostatic integrity or
thrombosis.
By the same token, antiplasmin levels can be assayed to evaluate
fibrinolytic function. For example, by adding a plasmin-containing
reagent to a hematological sample in a reaction tube, antiplasmin
can then be quantitated.
Furthermore, the blood coagulation system must be regulated to
prevent massive formation of thrombi. Blood consequently contains
natural clotting cascade inhibitors.
Protein C is a coagulation inhibitor, functioning to lock the
activity of activated Factor VIII, as well as another clotting
factor. At the same time, Protein C inactivates an inhibitor of
tissue plasminogen activator (tPA is secreted primarily by
endothelial cells and activates plasmin), thus enhancing
fibrinolytic activity.
Levels of Protein C can be assayed by adding to a sample a
chromogenic synthetic peptide substrate that is cleaved in the
presence of the reaction between a protease (Factor VIII, for
example) and its inhibitor (Protein C, for example). Cleavage of
the synthetic substrate produces a chromogenic change that can be
quantitated photometrically.
The intrinsic clotting pathway by definition can be activated
solely by elements within the blood itself, when intrinsic Factor
XII (Hageman Factor) comes into contact with and is bound by a
negatively charged surface (thus the pathway can be triggered in
vitro). The time to coagulation via this mechanism is referred to
as the "partial thromboplastin time," in relation to the time to
coagulation required when the extrinsic pathway is initiated via
thromboplastin.
The intrinsic clotting system can be screened generally for
abnormalities by the activated partial thromboplastin time (aPTT)
assay. This test is also used to monitor the anticoagulant effect
of heparin treatment. Heparin occurs naturally in the basophils of
blood leukocytes, but is prepared as a commercial product from
animal sources and administered therapeutically. Heparin
accelerates the activity of Antithrombin III (ATIII). ATIII is the
major inhibitor of the enzymes of the clotting cascade, binding to
some half-dozen proteases, including factors X and XII and
thrombin. Antithrombin III acting with co-factor heparin functions
immediately to inhibit coagulation.
The aPTT assay is conducted by adding a test reagent containing a
Factor XII activator to a platelet-poor plasma sample. Time to
clotting is evaluative of adequate levels of the intrinsic
coagulation factors.
Antithrombin III levels can be assayed by employing a synthetic
peptide substrate as with the assay for Protein C levels described
above.
In the above-described automated hematological analyzers, measuring
reagents and test samples are taken up by means of a pipette-like
aspirating device (sampling probe) and ejected into reaction
vessels.
Ideally, pipette devices used herein would be provided according to
use, as it were, for each particular assaying reagent or each
particular sample. However, due to cost and apparatus size
limitations, a single pipette device is utilized in common. Herein,
if the pipette device is not cleaned sufficiently the problem of
"carry-over" occurs, in which prior-remaining matter is mixed in
with next-aspirated reagents, exerting an influence on the
analytical results.
In hemostasis and thrombosis assays, methods include clotting time
and chromogenic substrate tests, as described above, as well as
immunoassays. Among the reagents employed in these assaying methods
are many that have enzymatic activity, or in the case of
chromogenic substrates, that contain proteins or peptides. Because
enzymes, and proteins and peptides generally, tend to become
adsorbed on the pipette device, carry-over problems are liable to
arise with the use of such reagents, making sufficient cleaning of
the pipette device necessary. Conventionally the pipette device has
been cleaned with a cleansing fluid containing a hypochlorite
substance.
Nevertheless, there are situations in which using the above-noted
cleansing fluid insufficiently cleans the pipette device in the
automated analyzer. This has been particularly so with the
foregoing plasmin-containing anti-plasmin assaying reagents, and PT
reagents containing recombinant tissue factor. Moreover, increasing
the concentration of the hypochlorite substance is not
satisfactory, since this damages the tubes and other fluid
components, or else brings about effects due to the residual
hypochlorite substance itself. Further, the undesirability of a
drop in processing capacity (throughput) of the automated analyzer
due to the cleaning process therefore demands instantaneous
cleansing.
SUMMARY OF THE INVENTION
An object of the present invention is to enable instantaneous
cleaning of a sampling probe in an automated analyzer, and at the
same time to curtail carry-over effects from the cleansing agent or
from assaying reagents residual in the sampling probe due to
insufficient cleaning.
Another object of the invention is to cleanse a pipette or sampling
probe employed in aspirating and dispensing aliquots of analytical
samples, assaying reagents, and reaction solutions thereof for
multiple assays in an automated analyzer, by a method of contacting
surfaces of the pipette or sampling probe at least subsequent to
each assay with a specially prepared cleansing composition, so as
to remove instantaneously assaying reagents such as enzymes,
proteins and peptides contaminating the surfaces of the pipette or
sampling probe, without contaminating the surfaces with the
cleansing composition itself.
The specially prepared cleansing composition is an acidic aqueous
solution of pH 5.0 or less; preferably the pH is about 1.8 to 4.0;
most preferably the pH is about 2.0 to 2.5. In order to maintain
acidic pH, adjustment can be made utilizing conventional acidifying
agents (hydrochloric acid, phosphoric acid, citric acid, for
example), and buffers (Goode buffers, for example). The amount of
acid added to he composition depends on the desired pH, and the
concentration and strength of the acid.
As a substance having a primary amino group, amino acids, and
"Tris" [tris(hydroxymethyl)aminomethane] or the like can be used.
The amino acids are not particularly limited, however glycine,
valine, leucine, phenylalanine, alanine, serine, and threonine are
preferable; glycine is most preferable. The concentration of the
compound having a primary amino group can be suitably determined
according to the compound employed; but for example, amino acids
can be employed in a concentration in the range of 0.05 to 10 w/v
%, more preferably in the range of 0.1 to 2.0 w/v %.
Furthermore, a small amount of nonionic surfactant elevates the
cleansing effectiveness of the composition. For example,
polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers,
polyoxyethylene sorbitan esters, polyoxyethylene alkyl esters, and
mixtures thereof can be used. If the nonionic surfactant is water
soluble, the ethylene oxide or polyoxyethylene molar addition
number is not particularly limited; however 2 to 100 moles is
preferable, 5 to 60 moles is more preferable, and 5 to 12 moles is
most preferable. The cleansing effectiveness is elevated further by
mixing a nonionic surfactant of low ethylene oxide molar addition
number with one of high ethylene oxide molar addition number.
The polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether
and polyoxyethylene alkyl ester include an alkyl group having 8 to
20 carbons, and preferably an alkyl group having 8 to 18
carbons.
Several commercially produced nonionic surfactants suitable for use
in the specially prepared composition are available. For example,
polyoxyethylene alkyl ether in several ethylene oxide molar
addition numbers is available as Actinol L.TM. (Matsumoto Yushi
Pharmaceuticals, Inc.), and Noigen ET.TM. (Daiichi Kogyo
Pharmaceuticals, Inc.); polyoxyethylene alkyl phenyl ether in
several ethylene oxide molar addition numbers is available as
Emulsit.TM. (Daiichi Kogyo Pharmaceuticals, Inc.), and Nonipol.TM.
(Sanyo Kasei Kogyo, Inc.); polyoxyethylene sorbitan ester in
several ethylene oxide molar addition numbers is available as
Span.TM. (Atlas Powder Co.), and Solubon.TM. (Toho Kagaku Kogyo,
Inc); polyoxyethylene alkyl ester in several ethylene oxide molar
addition numbers is available as Noigen ES.TM. (Daiichi Kogyo
Pharmaceuticals, Inc). Furthermore, the concentration of the
nonionic surfactant can be suitably determined according to the
employed surfactant; but the surfactant can be employed in the
range of 0.001 to 2.0 w/v %, and more preferably in the range of
0.10 to 1.00 w/v %.
The cleansing method of the present invention is extremely
effective in instantaneously cleansing the quantitatively
aspirating pipette or sampling probe wherein concurrent multi-item
analysis is conducted by an automated analyzer having a liquid
dispensing system.
After the sampling probe dispenses a reagent in the process of
assaying one test item, the aspirating probe which has
aspirated/ejected sample aliquots and reagents, or their reaction
solution, is cleansed with the specially prepared cleansing
composition, and a rinse is performed as desired. The pipette being
cleansed draws in an amount of the cleansing composition sufficient
to contact substantially all contaminated surfaces thereof and then
discharges the spent cleansing composition to a suitable waste
site. Optionally the pipette can be rinsed with a conventional
rinse solution, such as water. Subsequently analysis is carried out
on another assay item. It is sufficient to have an extremely short
period for the exposure of the aspirating probe to the cleansing
composition. The preferable duration of the exposure is about 0.1
to 20 seconds, and more preferably about 0.2 to 1.0 seconds. The
specially prepared cleansing composition may be used in combination
with other types of cleansing compositions, for example, with
hypochlorite cleansing compositions.
As an example of a preferred chemical form for the specially
prepared cleansing composition, hydrochloric acid glycine
(polyoxyethylene).sub.n nonyl phenyl ether can be given. Glycine
can be replaced with valine, leucine, phenylalanine, alanine,
serine, or threonine. Furthermore, hydrochloric acid can be
replaced with acetic acid or citric acid.
The cleansing composition in the method according to the present
invention is superior in preventing carry-over of reagents
remaining in the pipette or sampling probe wherein multiple assays
including those having enzymatic activity, or employing proteins or
the peptides of chromogenic substrates, are carried out
continuously, such as is the case with automated devices for
carrying out hemostasis or thrombosis analysis.
The foregoing and other objects, features, aspects and advantages
of the present invention will become more apparent from the
following detailed description, illustrated by way of the following
examples.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is an exploded, oblique view schematically illustrating an
automated hematological analyzer, in which a method in accordance
with the present invention may be utilized.
DETAILED DESCRIPTION OF THE INVENTION
Analysis flow in an automated hematological analyzer will be
described with reference to FIG. 1. Centrifuged blood sample tubes
are arranged in a sample rack 1. When the analytical procedure
begins, requisite sample aliquots are aspirated from the sample
tubes by sampling arm 2 and dispensed into a main reagent
refrigeration unit 3, wherein the samples and reagents are kept
refrigerated to suppress degradation. Next, for each assay item,
sampling arm 4 aspirates and dispenses a sample aliquot, which is
then incubated in a sample-heating unit 5. After a fixed time, an
assaying reagent is dispensed with reagent-aspirating pipette
(quantitative sampling probe) arm 6 (in the figure, the thick
pipette 6a has a heater, and the slender pipette 6b is exclusively
for thrombin) into the incubated sample aliquot. Herein, XY
mechanism 7 functions as a stirrer to mix the reagents and samples
sufficiently. XY mechanism 7 then transports the samples to a
(not-shown) photometric unit. The assayed samples are then disposed
of, again by XY mechanism 7, after which the analytical procedure
is complete.
EXAMPLE 1
______________________________________ Cleansing Preparation
Composition I ______________________________________ glycine 0.25
w/v % 6N hydrochloric acid 0.31 v/v % Emulsit 16
[(polyoxyethylene).sub.n variable conc. nonyl phenyl ether, n = 40]
pH 2.2 ______________________________________
Demonstrated Cleansing Effectiveness Test Data
Cleansing Effectiveness Depending on Difference in Concentration of
"Emulsit 16"
Procedure
In an Automated Blood Coagulation Analyzer CA-6000, manufactured by
Toa Medical Electronics Co., Ltd., highly abnormal prothrombin time
(PT) plasma sample aliquots were initially assayed with Dade Co.'s
"Innovin," a PT assaying reagent containing recombinant tissue
factor. Then, utilizing a Factor VIII quantitating reagent, Factor
VIII quantitation of further sample aliquots was conducted. In
these analyses, clotting time was obtained by photometrically
detecting, as a change in intensity of diffused light, the change
in turbidity due to the fibrin clot that arises when sample and
reagent are mixed. Following the analysis with "Innovin," the
degree of the carry-over from the PT reagent to the Factor VIII
quantitating reagent was examined (a) wherein the
reagent-aspirating pipette was not cleaned, (b) wherein the
reagent-aspirating pipette was cleaned utilizing a conventional
cleansing composition having a hypochlorite concentration of about
1.0 w/v %, and (c) wherein the reagent-aspirating pipette was
cleansed utilizing cleaning solutions which were prepared by
varying the concentration of "Emulsit 16" in the above Composition
I.
For each of these respective cases, the resultant clotting times in
the Factor VIII assay, as well as activeness percentages are
indicated below. The activeness percentages of the samples were
calculated from a previously obtained calibration curve relating
clotting time and activeness percentage. The result of Factor VIII
quantitation independently without assaying with "Innovin" was used
as the control value.
______________________________________ Results Factor VIII
Quantitation Clotting Activeness Cleansing Method Time Percentage
______________________________________ Control 125.4 s 0.4% a)
Reagent pipette not 50.2 s 559.2% cleansed b) Cleansed with hypo-
72.0 s 54.2% chlorite cleansing agent c) Cleansed with Composition
I using Emulsit 16, changing concentrations Emulsit 16 0.01 w/v %
78.8 s 30.2% Emulsit 16 0.10 w/v % 105.9 s 2.7% Emulsit 16 0.25 w/v
% 109.9 s 1.9% Emulsit 16 0.50 w/v % 110.0 s 1.9%
______________________________________
With the clotting time control measurement in the Factor VIII assay
125.4 sec., a tendency to extreme prolongation was indicated,
against which a
tendency to shortening was indicated, due to carry-over of the PT
reagent, (a) wherein the pipette was not cleaned, and (b) wherein
it was cleaned utilizing the conventional hypochlorite cleansing
composition, with clotting times in the Factor VIII assay 50.2 sec.
and 72.0 sec., respectively.
On the other hand, (c) wherein the pipette was cleansed with
cleansing solutions prepared from Composition I, changing
concentrations of "Emulsit 16," with a 0.10 w/v % concentration or
more, a tendency to prolongation in clotting times in the Factor
VIII assay was indicated. Comparing the activeness percentages with
the value of the control measurement also yields similar results.
In short, carry-over of the PT reagent could be prevented.
EXAMPLE 2
______________________________________ Cleansing Preparation
Composition II ______________________________________ glycine 0.25
w/v % 6N hydrochloric acid 0.31 v/v % Emulsit
[(polyoxyethylene).sub.n nonyl phenyl ether, 0.25 w/v % variable
molar addition number] pH 2.2
______________________________________
Demonstrated Cleansing Effectiveness Test Data
Cleansing Effectiveness Depending on Difference in Polyoxyethylene
Molar Addition Number in "Emulsit"
Procedure
In the Automated Blood Coagulation Analyzer CA-6000, highly
abnormal prothrombin time (PT) plasma sample aliquots were
initially assayed with "Innovin." Then, utilizing a Factor VIII
quantitating reagent, Factor VIII quantitation of further sample
aliquots was conducted. In these analyses, clotting time was
obtained by photometrically detecting as a change in intensity of
diffused light the change in turbidity due to the fibrin clot that
arises when sample and reagent are mixed. Following the analysis
with "Innovin," the degree of the carry-over from the PT reagent to
the Factor VIII quantitating reagent was examined (a) wherein the
reagent-aspirating pipette was not cleaned, (b) wherein the
reagent-aspirating pipette was cleaned utilizing a conventional
cleansing composition having a hypochlorite concentration of about
1.0 w/v % and (c) wherein the reagent-aspirating pipette was
cleansed utilizing cleaning solutions which were prepared by
varying polyoxyethylene molar addition numbers in "Emulsit" in the
above Composition II.
For each of these respective cases, the resultant clotting times in
the Factor VIII assay, as well as activeness percentages are
indicated below. The result of Factor VIII quantitation
independently without assaying with "Innovin" was used as the
control value.
______________________________________ Results Factor VIII
Quantitation Clotting Activeness Cleansing Method Time Percentage
______________________________________ Control 125.4 s 0.4% a)
Reagent pipette not 50.2 s 559.2% cleansed b) Cleansed with hypo-
72.0 s 54.2% chlorite cleansing agent c) Cleansed with Composition
II using Emulsit, changing polyoxyethylene molar addition number
Emulsit 9 (n = 30) 0.25 w/v % 112.3 s 1.5% Emulsit 16 (n = 40) 0.25
w/v % 110.2 s 1.8% Emulsit 25 (n = 50) 0.25 w/v % 100.6 s 4.3%
Emulsit 100 (n = 100) 0.25 w/v % 85.2 s 17.0%
______________________________________
With the clotting time control measurement in the Factor VIII assay
125.4 sec., a tendency to extreme prolongation was indicated,
against which a tendency to shortening was indicated, due to
carry-over of the PT reagent, (a) wherein the pipette was not
cleaned, and (b) wherein it was cleaned utilizing the conventional
hypochlorite cleansing composition, with clotting times in the
Factor VIII assay 50.2 sec. and 72.0 sec., respectively.
On the other hand, (c) wherein the pipette was cleansed with
cleansing solutions prepared from Composition II, changing
polyoxyethylene molar addition numbers in "Emulsit," with the
addition number n=50 or less, a tendency to prolongation in
clotting times in the Factor VIII assay was indicated. Comparing
the activeness percentages with the value of the control
measurement also yields similar results. In short, carry-over of
the PT reagent could be prevented.
EXAMPLE 3 ______________________________________ Cleansing
Preparation Composition III ______________________________________
glycine 0.25 w/v % 6N hydrochloric acid 0.31 v/v % Emulsit 16
[(polyoxyethylene).sub.n nonyl phenyl 0.25 w/v % ether, n = 40]
Nonipol [(polyoxyethylene).sub.n nonyl phenyl ether, 0.25 w/v %
variable molar addition number] pH 2.2
______________________________________
Demonstrated Cleansing Effectiveness Test Data
Cleansing Effectiveness Depending on Supplementation of Non-Ionic
Surfactant of Low Polyoxyethylene Molar Addition Number
Procedure
In the Automated Blood Coagulation Analyzer CA-6000, highly
abnormal prothrombin time (PT) plasma sample aliquots were
initially assayed with "Innovin." Then, utilizing a Factor VIII
quantitating reagent, Factor VIII quantitation of further sample
aliquots was conducted. In these analyses, clotting time was
obtained by photometrically detecting as a change in intensity of
diffused light the change in turbidity due to the fibrin clot that
arises when sample and reagent are mixed. Following the analysis
with "Innovin," the degree of carry-over from the PT reagent to the
Factor VIII quantitating reagent was examined (a) wherein the
reagent-aspirating pipette was not cleaned, (b) wherein the
reagent-aspirating pipette was cleaned utilizing a conventional
cleansing composition having a hypochlorite concentration of about
1.0 w/v % and (c) wherein the reagent-aspirating pipette was
cleaned utilizing cleaning solutions prepared supplementing
non-ionic surfactants of low polyoxyethylene molar addition number
in cleansing compositions from the above Composition III.
For each of these respective cases, the resultant clotting times in
the Factor VIII assay, as well as activeness percentages are
indicated below. The result of Factor VIII quantitation
independently without assaying with "Innovin" was used as the
control value.
______________________________________ Results Factor VIII
Quantitation Clotting Activeness Cleansing Method Time Percentage
______________________________________ Control 128.1 s 0.3% a)
Reagent pipette not 51.2 s 492.2% cleansed b) Cleansed with
hypo-chlorite 73.5 s 47.4% cleansing agent c) Cleansed with
Composition III including supplemental non-ionic surfactants of low
polyoxy- ethylene molar addition number Nonipol 55 (n = 5.5) 0.25
w/v % 126.1 s 0.4% Nonipol 70 (n = 7) 0.25 w/v % 128.3 0.3% Nonipol
90 (n = 9) 0.25 w/v % 125.7 s 0.4% Nonipol 100 (n = 10) 0.25 w/v %
126.6 s 0.4% Nonipol 120 (n = 12) 0.25 w/v % 128.1 s 0.3%
______________________________________
With the clotting time control measurement in the Factor VIII assay
128.1 sec., a tendency to extreme prolongation was indicated,
against which a tendency to shortening was indicated, due to
carry-over of the PT reagent, (a) wherein the pipette was not
cleaned, and (b) wherein it was cleaned utilizing the conventional
hypochlorite cleansing composition, with clotting times in the
Factor VIII assay 51.2 sec. and 73.5 sec., respectively.
On the other hand, (c) wherein the pipette was cleansed with
cleansing solutions prepared from Composition III, supplementing
non-ionic surfactants of low polyoxyethylene molar addition number,
n=5.5 to 12, a tendency to prolongation in clotting times in the
Factor VIII assay was indicated. Comparing the activeness
percentages with the value of the control measurement also yields
similar results. In short, carry-over of the PT reagent could be
prevented.
EXAMPLE 4
______________________________________ Cleansing Preparation
Composition IV ______________________________________ glycine 0.25
w/v % 6N hydrochloric acid 0.31 v/v % Emulsit 16
[(polyoxyethylene).sub.n nonyl phenyl 0.25 w/v % ether, n = 40]
Nonipol [(polyoxyethylene).sub.n nonyl phenyl ether, 0.25 w/v % n =
7] pH variable ______________________________________
Demonstrated Cleansing Effectiveness Test Data
Cleansing Effectiveness Depending on Cleaning Solution pH
Procedure
In the Automated Blood Coagulation Analyzer CA-6000, anti-plasmin
in sample aliquots of normal human plasma was initially assayed
utilizing an anti-plasmin assaying reagent containing plasmin.
Then, a Protein C activity analysis of sample aliquots was
conducted employing a chromogenic substrate procedure. Following
assay by the anti-plasmin reagent, the degree of the carry-over of
the anti-plasmin reagent to Protein C activity analysis was
examined (a) wherein the reagent-aspirating pipette was not
cleaned, (b) wherein the reagent-aspirating pipette was cleaned
utilizing a cleansing composition having a hypochlorite
concentration of about 1.0 w/v % and (c) wherein the
reagent-aspirating pipette was cleaned utilizing cleaning solutions
prepared varying the pH in cleansing compositions from the above
Composition IV by adding 1N NaOH accordingly.
For each of these respective cases, the Protein C activeness
percentages are indicated below. The result for Protein C
activeness independently without assaying with antiplasmin was used
as the control value.
______________________________________ Results Cleansing Method
Protein C Activeness ______________________________________ Control
102.5% a) Reagent pipette not cleansed 160.0% b) Cleansed with
hypo-chlorite 152.0% cleansing agent c) Cleansed with Composition
IV, changing pH of cleaning solution pH 2.0 103.5% pH 4.0 101.2% pH
5.0 108.0% pH 9.0 125.8% pH 12.0 138.9%
______________________________________
In the control measurement, Protein C activeness displayed a normal
value of 102.5%, against which (a) wherein the pipette was not
cleaned, and (b) wherein it was cleaned utilizing the conventional
hypochlorite cleansing composition, a tendency to abnormally high
Protein C activeness values, 160.0% and 152.0%, respectively, was
exhibited, due to carrying-over of antiplasmin reagent.
On the other hand, (c) wherein the pipette was cleansed with
cleansing solutions prepared from Composition IV, changing the pH,
from pH 5.0 to 2.0, Protein C activeness normalized, yielding
results similar to the value of the control measurement. In short,
carry-over of the anti-plasmin reagent could be prevented.
EXAMPLE 5
______________________________________ Cleansing Preparation
Composition V ______________________________________ glycine 0.25
w/v % 6N hydrochloric acid 0.31 v/v % Emulsit 16
[(polyoxyethylene).sub.n nonyl phenyl 0.25 w/v % ether, n = 40]
Nonipol [(polyoxyethylene).sub.n nonyl phenyl ether, 0.25 w/v % n =
7] pH 2.2 ______________________________________
Demonstrated Cleansing Effectiveness Test Data
Effectiveness of Cleansing Solution in Preventing Carry-Over in
Various Assays
Procedure
In the Automated Blood Coagulation Analyzer CA-6000, utilizing
reagents (A) in inducing carry-over and reagents (B) to incur
carry-over, concurrent two-item assays were conducted. Following
assay utilizing the reagent for inducing carry-over, (a) wherein
the reagent-aspirating pipette was not cleaned, and (b) wherein the
pipette was cleaned utilizing a cleansing composition from the
above Composition V, the degree of carry-over from
reagent (A) to reagent (B) was examined. The result assayed
independently with reagent (B) was taken as the control value.
______________________________________ Results Without With Reagent
A Reagent B Sample Control Cleansing Cleansing
______________________________________ Recombinant aPTT Heparin-
118.3 s 70.3 s 118.0 s PT Added Plasma Recombinant Factor VIII
Normal 100.0% 595.0% 99.8% PT Human Plasma Anti- aPTT Normal 31.7 s
36.6 s 31.8 s plasmin Human Plasma Anti- Protein C Normal 102.0%
163.0% 102.5% plasmin Human Plasma ATIII aPTT Normal 31.6 s 38.3 s
31.5 s Human Plasma ATIII Protein C Normal 102.0% 50.0% 103.5%
Human Plasma ______________________________________
Comparing against the control value the results from the
measurements carried out without cleansing the reagent-aspirating
pipette between assays demonstrates striking differences. In
contrast, cleansing the pipette between assays yielded results
similar to the value of the control measurement. In short,
carry-over of reagent A to reagent B could be prevented by a
cleansing composition from Composition V.
A cleansing method in accordance with the present invention
practically eliminates carry-over, securing accuracy of analytical
results, particularly in hemostatic and thrombotic assays employing
enzymatically active or peptidyl reagents.
Various details of the present invention may be changed without
departing from its spirit nor its scope. Furthermore, the foregoing
description of the embodiments according to the present invention
are provided for illustration only, and not for the purpose of
limiting the invention as defined by the appended claims and their
equivalents.
* * * * *